There is considerable interest by biochemists, clinical chemists and pharmaceutical manufacturers in determining the concentration of amino acids, peptides and other amine-functional compounds in a complex biological sample. This analysis may require, for example, the determination of twenty or more amino acids at the picomole level. A typical application is the qualitative and quantitive analysis of the amino acids that are present in a synthetic peptide, perhaps one which has been synthesized by recombinant DNA methods. In this application, it is not uncommon for the analyst to be forced to work with sub-microgram quantities of a sample. Similarly, the analysis for amino acids in the blood of neonates, for example, demands the handling of very small samples.
In order to reduce the sample size to accomodate the potential scarcity of sample available and provide detection capability in the presence of non-amino acid components, the analytical method that is selected must provide a very high degree of sensitivity and detection selectivity. The sensitivity and selectivity of an assay for a compound is dependent on the instrumental response that it provides. Typical detection schemes involve the absorbance or emission of light, or the electrochemical reactivity of the compound(s) of interest.
The qualitative and quantitative analysis of complex mixtures of amino acids and/or peptides involves analyzing samples that may contain twenty or more amino acids, and in which the structural differences between many of the amino acids are subtle, e.g., the difference between leucine and valine is one methylene group in the aliphatic side chain. Thus, a high degree of selectivity is needed to distinguish them. A typical mixture contains amino acids with acidic, basic and neutral side groups.
The absorbance, fluorescence and electrochemical response of most amino acids is quite weak. A commonly used tactic to maximize the sensitivity of an assay is to convert the compound(s) of interest into a derivative that exhibits a better response than the compound of interest itself. The selection of a derivatizing agent is a critical choice in the development of an analytical procedure. The derivatizing agent affects the ultimate sensitivity and accuracy of the analysis by maximizing the sensitivity, yield and stability of the derivatized amino acids.
There are several criteria important for the utility of an derivatization method. The analytical procedure must provide accurate quantitation for each component present in a complex mixture. To accomplish this, it is necessary to resolve the components of interest, not only from each other, but from components generated by the derivatization procedure. Quantitative conversion of all underivatized amino acids, including secondary amino acids, to single products is highly desirable, and facilitates good quantitation.
Detection selectivity is another advantageous feature for amino acid derivatives. Underivatized amino acids all absorb weakly in the low UV (200-220 nm) range, but detection at such wavelengths is subject to interference by many compounds present in sample mixtures or chromatographic mobile phases. Derivatization with reagents absorbing at approximately 254 nm provides a measure of selectivity, but any aromatic organic compounds, frequently present in biological samples, can interfere at this wavelength. Reagents that enable detection via fluorescence, electrochemical response or visible-range absorbance would be desirable for superior detection selectivity.
Finally, it is necessary for derivatives to be sufficiently stable to allow separation and detection without significant degradation. Highly stable derivatives are also favorable as they allow a sample to be reanalyzed, if so desired, without assaying another sample.
In the past, a number of derivatization procedures have been developed to permit the assay of amino acids by high performance liquid chromatographic and electrophoretic separations. Four such procedures commonly utilized for this purpose include:
1) The o-phthalaldehyde (OPA)/mercaptan method. The OPA procedure can detect amino acids with a typical detectable level in the order of about 100 femtomole (fmol). The formation of the derivatives is rapid. A significant difficulty with this method is that the adduct is fairly unstable, and must be prepared very shortly before the detection step. An additional problem is that this reagent will not form a derivative with secondary amino acids.
2) The 9-fluorenylmethylchloroformate (FMOC method). The FMOC procedure provides for stable derivatives, with a minimum detectable level in the order of a few hundred fmol. There are a number of disadvantages with the FMOC procedure. Free tryptophan and cystine cannot be quantitated easily. The derivatizing reagent must be removed from the reaction mixture by an extraction step because it is itself fluorescent. The reagent has also been reported to form multiple derivatives with histidine. The reagent is also hazardous to work with, because it is corrosive and is a lachrymator.
3) The phenylisothiocyanate method (PITC). The PITC procedure yields stable derivatives which are formed rapidly. It can be used for both primary and secondary amino acids, as well as cystine. The method uses absorbance as the detection procedure, and can provide a minimum detection limit of 1 pmol. However, the derivatives are not fluorescent and detection must be performed at 254 nm, which does not allow for good detection selectivity.
4) The dansyl chloride method. The dansyl chloride method provides stable derivatives with a minimum detectability in the order of about 1.5 pmol. It is able to detect secondary amines and cystine, but it results in multiple derivatives.
In addition to the above methods, fluorescent succinimidocarbamates have been used as derivatizing agents for amines, amino acids, peptides, phosphates and other classes of compounds. When the succinimidocarbamate reagent is used to tag a compound with a fluorescent group, a detection limit of about 1 pmol can be achieved. These reagents are used in conjunction with modern separation techniques such as high performance liquid chromatography, thin layer chromatography or capillary electrophoresis. Nimura et al., Anal. Chem., 58:2372-2375 (1986). Succinimidyl activated carbamates have been prepared by reacting carbocyclic aromatic amines with di-(N-succinimidyl) carbonate. Takeda et al., Tetrahedron Lett., 24:4569-4572 (1983).
Improved methods for detecting and accurately measuring the presence and amount of amino-functional compounds, particularly amino acids and proteins, are needed. A method which allows detection of these compounds in subpicomole quantities in very small samples and which minimizes the amount of sample that is consumed in the analysis, is needed.